Cloning and characterization of fiber type-specific ryanodine receptor isoforms in skeletal muscles of fish

We have cloned agroup of cDNAs that encodes the skeletal ryanodine receptor isoform(RyR1) of fish from a blue marlin extraocular muscle library. The cDNAsencode a protein of 5,081 amino acids with a calculated molecular massof 576,302 Da. The deduced amino acid sequence shows strong sequenceidentity to previously characterized RyR1 isoforms. An RNA probederived from a clone of the full-length marlin RyR1 isoform hybridizesto RNA preparations from extraocular muscle and slow-twitch skeletalmuscle but not to RNA preparations from fast-twitch skeletal or cardiacmuscle. We have also isolated a partial RyR clone from marlin andtoadfish fast-twitch muscles that shares 80% sequence identity withthe corresponding region of the full-length RyR1 isoform, and a RNAprobe derived from this clone hybridizes to RNA preparations fromfast-twitch muscle but not to slow-twitch muscle preparations. Westernblot analysis of slow-twitch muscles in fish indicates the presence ofonly a single high-molecular-mass RyR proteincorresponding to RyR1. [³H]ryanodine bindingassays revealed the fish slow-twitch muscle RyR1 had a greatersensitivity for Ca²⁺ than thefast-twitch muscle RyR1. The results indicate that, in fish muscle,fiber type-specific RyR1 isoforms are expressed and the two proteinsare physiologically distinct.

     

Raccoons (Procyon lotor) in Germany as potential reservoir species for Lyssaviruses

Raccoons can be found almost everywhere in Germany since their first successful introduction in 1934. Although the animal is a well-known reservoir species for rabies in the USA, during the last European fox rabies epizootic, only a few rabid raccoons were reported from Germany. In recent years, the raccoon population density has increased tremendously, especially in (semi) urban settings. Presently, Germany is free of terrestrial wildlife rabies. To assess the potential risk that the raccoon population in Germany could act as a reservoir species upon reemergence of rabies, the susceptibility of the local raccoon population was investigated. Wild-caught animals were inoculated with the most likely lyssavirus variants to infect the local population. It was shown that the raccoons were fully susceptible for a dog and raccoon rabies virus isolate. Also, five of six raccoons inoculated with a fox rabies virus isolate showed clinical signs. However, none of the raccoons infected with European Bat Lyssavirus type 1 succumbed to rabies; meanwhile, all these raccoons seroconverted. It is concluded that the highest risk for the raccoon population in Germany to become infected with lyssaviruses is through the importation of rabies infected dogs.     

Diffusional mobility of parvalbumin in spiny dendrites of cerebellar Purkinje neurons quantified by fluorescence recovery after photobleaching

Ca(2+)-binding proteins (CaBPs) represent key factors for the modulation of cellular Ca(2+) dynamics. Especially in thin extensions of nerve cells, Ca(2+) binding and buffered diffusion of Ca(2+) by CaBPs is assumed to effectively control the spatio-temporal extend of Ca(2+) signals. However, no quantitative data about the mobility of specific CaBPs in the neuronal cytosol are available. We quantified the diffusion of the endogenous CaPB parvalbumin (PV) in spiny dendrites of cerebellar Purkinje neurons with two-photon fluorescence recovery after photobleaching. Fluorescently labeled PV diffused readily between spines and dendrites with a median time constant of 49 ms (37-61 ms, interquartile range). Based on published data on spine geometry, this value corresponds to an apparent diffusion coefficient of 43 microm(2) s(-1) (34-56 microm(2) s(-1)). The absence of large or immobile binding partners for PV was confirmed in PV null-mutant mice. Our data validate the common but so far unproven assumption that PV is highly mobile in neurons and will facilitate simulations of neuronal Ca(2+) buffering. Our experimental approach represents a versatile tool for quantifying the mobility of proteins in neuronal dendrites.     

Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine,l-Valine,and 2-Ketoisovalerate

Exchange of the native Corynebacterium glutamicum promoter of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutated dapA promoter variants led to a series of C. glutamicum strains with gradually reduced growth rates and PDHC activities. Upon overexpression of the l-valine biosynthetic genes ilvBNCE, all strains produced l-valine. Among these strains, C. glutamicum aceE A16 (pJC4 ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of the pqo and ppc genes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities, C. glutamicum aceE A16 Δpqo Δppc (pJC4 ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter) l-valine with an overall yield (Y_P/S) of 0.36 mol per mol of glucose and a volumetric productivity (Q_P) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression of ilvBNCD instead of ilvBNCE transformed the l-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with a Y_P/S of 0.24 mol per mol of glucose and a Q_P of 6.9 mM per h [0.8 g/(liter × h)]. The replacement of the aceE promoter by the dapA-A16 promoter in the two C. glutamicum l-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate that C. glutamicum strains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.     

Comparing tandem repeats with duplications and excisions of variable degree

Traditional sequence comparison by alignment employs a mutation model comprised of two events, substitutions and indels (insertions or deletions) of single positions. However, modern genetic analysis knows a variety of more complex mutation events (e.g., duplications, excisions, and rearrangements), especially regarding DNA. With ever more DNA sequence data becoming available, the need to accurately compare sequences which have clearly undergone more complicated types of mutational processes is becoming critical. Herein we introduce a new method for pairwise alignment and comparison of sequences with respect to the special evolution of tandem repeats: substitutions and indels of single positions and, additionally, duplications and excisions of variable degree (i.e., of one or more repeat copies simultaneously) are taken into account. To evaluate our method, we apply it to the spa VNTR (variable number of tandem repeats) cluster of Staphylococcus aureus, a bacterium of high medical importance     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

Protein identification and tracking in two-dimensional electrophoretic gels by minimal protein identifiers

Protein identification by matrix-assisted laser desorption/ionization mass-spectrometry peptide mass fingerprinting (MALDI-MS PMF) represents a cornerstone of proteomics. However, it often fails to identify low-molecular-mass proteins, protein fragments, and protein mixtures reliably. To overcome these limitations, PMF can be complemented by tandem mass spectrometry and other search strategies for unambiguous protein identification. The present study explores the advantages of using a MALDI-MS-based approach, designated minimal protein identifier (MPI) approach, for protein identification. This is illustrated for culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv after separation by two-dimensional gel electrophoresis (2-DE). The MPI approach takes into consideration that proteins yield characteristic peptides upon proteolytic cleavage. In this study, peptide mixtures derived from tryptic protein cleavage were analyzed by MALDI-MS and the resulting spectra were compared with template spectra of previously identified counterparts. The MPI approach allowed protein identification by few protein-specific signature peptide masses and revealed truncated variants of mycobacterial elongation factor EF-Tu, previously not identified by PMF. Furthermore, the MPI approach can be employed to track proteins in 2-DE gels, as demonstrated for the 14 kDa antigen, the 10 kDa chaperone, and the conserved hypothetical protein Rv0569 of M. tuberculosis H37Rv. Furthermore, it is shown that the power of the MPI approach strongly depends on distinct factors, most notably on the complexity of the proteome analyzed and accuracy of the mass spectrometer used for peptide mass determination.     

The ubiquitin C‐terminal hydrolase UCH‐L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C‐terminal hydrolase UCH‐L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH‐L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH‐L1‐negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH‐L1 controls the early membrane‐associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c‐cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2‐ and AKT‐dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH‐L1_C90S. These findings highlight a previously unrecognized involvement of the ubiquitin cycle in bacterial entry. UCH‐L1 is highly expressed in malignant cells that may therefore be particularly susceptible to invasion by bacteria‐based drug delivery systems.